KR100398065B1 - A new fungal strain Acremoniem sp. MT70646(KCTC 8973P), novel compounds produced by this strain and their use - Google Patents

Abstract

The present invention relates to a new strain of fungus Acremonium sp. MT70646 (KCTC 8973P) isolated from soil, and to the use of new compounds and new compounds produced from the strain, and more particularly, to the development of cancer cells during cancer metastasis. A new fungal acromoni that produces a substance that inhibits the heparanase and heparanase activity required for endothelial cell invasion or angiogenesis and inhibits metastasis of cancer cells. Acremonium sp. MT70646 (KCTC 8973P) and novel compounds isolated and purified from the new strain culture medium, and heparanase activity inhibitors, heparanase activity inhibitors, cancer metastasis inhibitors and blood vessels containing these new compounds as active ingredients It relates to a production inhibitor.

Description

New strain of fungi Acremoniium genus MCT70646 (BTCC 8973P), and novel compounds produced from the strain and its use {A new fungal strain Acremoniem sp. MT70646 (KCTC 8973P), novel compounds produced by this strain and their use}

The present invention relates to a new strain of fungus Acremonium sp. MT70646 (KCTC 8973P) isolated from soil, and to the use of new compounds and new compounds produced from the strain, and more particularly, to the development of cancer cells during cancer metastasis. Acremonium sp. MT70646 (KCTC 8973P), which inhibits the activity of heparanase and heparanase necessary for endothelial cell invasion or small blood vessel formation and produces substances that can inhibit the metastasis of cancer cells. And heparanase activity inhibitors, heparanase activity inhibitors, cancer metastasis inhibitors, and angiogenesis inhibitors.

In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group.

Tumor invasion or metastasis of cancer cells in mammalian cells involve enzymatic degradation of basement membranes outside the cell. Heparan sulfate or heparin, together with collagen, laminin and fibronectin, is one of the major components of the basement membrane present in most mammalian cells. Heparansulfate and heparin are complex linear polysaccharides in which disaccharide units of highly N-acetylated or N-sulfated glucosamine and hexuronate are continuously combined. Structurally very similar to each other.

Heparansepatate and heparin are degraded by heparanase and heparanase enzymes, respectively, where heparinase can degrade not only heparin but also heparasulfate. As early as these enzymes are known to be closely involved in angiogenesis or cancer metastasis processes [ J. Biol. Chem . 257, 2678-2686 (1982); Science 220, 313-325 (1983). In addition, inhibitors of these enzymes have been reported to inhibit cancer metastasis [ Biochemistry . 25, 5322-5328, (1986); Cancer Res . 50, 3631 ~ 3637, (1990)] The possibility of development as an anticancer agent has been suggested, and some researchers are searching for a new inhibitor. In addition, for cancer metastasis, new blood vessels are formed, through which the supply of nutrients is made and growth of cancer tissue occurs. Such angiogenesis requires vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). These growth factors are bound to heparin or heparin sulphate and are liberated when jellyfish or heparanase breaks them down and induces angiogenesis by inducing growth of angiogenic cells. Thus, inhibition of heparanase or heparanase activity can inhibit neovascular production and inhibit growth of cancer cells, in particular, growth of metastatic cancer cells [ J. Biol. Chem . 270, 11322-11326 (1995). As a method of measuring neovascular formation, many methods using vascular endothelial cells called "HUVEC" are known [ Biol. Pharm. Bull . 20, 1131-1135 (1997). To date, trachyspic acid ( J. Antibiotics 48, 357-362, (1994)) and A-72363C [ J. Antibiotics 49, 61-64 (1996)] have been reported as heparanase inhibitors. Suramin, already used clinically as an anticancer agent [ J. Biol. Chem . 266, 9661 to 9666 (1991), are also known to inhibit heparanase activity and to inhibit tumor cell metastasis.

Therefore, there is a need for the development of novel compounds capable of inhibiting heparanase or heparanase enzyme activity in small amounts, as well as inhibiting metastasis and angiogenesis by inhibiting them in cells.

The present inventors have conducted a long time research to develop a substance showing a strong inhibitory activity against heparanase as part of the development of a new type of anticancer agent. As a result, the fungus strain Acremoniium MT70646 (KCTC 8973P) was isolated and separated from the soil, and the novel compound represented by the formula (1) that strongly inhibits heparanase and heparanase from the culture medium of this strain was separated and extracted. Was completed.

Therefore, the present invention is a novel fungal strain Acremoniium MT70646 (KCTC 8973P) isolated from the soil, a novel compound represented by the formula (1) extracted from the new strain culture, and heparina containing the new compound as an active ingredient It is an object of the present invention to provide inhibitors, heparanase inhibitors, cancer metastasis inhibitors and angiogenesis inhibitors.

1 shows a proton nuclear magnetic resonance spectrum of a compound represented by Chemical Formula 1a (CRM646-A),

2 shows carbon nuclear magnetic resonance spectra of the compound represented by Chemical Formula 1a (CRM646-A).

3 shows the HMBC nuclear magnetic resonance spectrum of the compound represented by Formula 1a (CRM646-A),

4 shows a proton nuclear magnetic resonance spectrum of a compound represented by Chemical Formula 1b (CRM646-B),

5 shows carbon nuclear magnetic resonance spectra of the compound represented by Chemical Formula 1b (CRM646-B),

Figure 6 shows the HPLC spectrum of the derivatives produced by the strain mycorrhiza fungi Acremoniium MT70646 (KCTC 8973P).

Figure 7 shows the inhibitory activity of metastasis (cancer cell infiltration) of B16 melanoma cells through the synthetic base membrane of derivatives produced by the mycobacteria fungus Acremoniium genus MT70646 (KCTC 8973P).

The present invention is characterized by the new strain Acremonium genus MT70646 (KCTC 8973P).

In another aspect, the present invention is characterized by another compound represented by the following formula (1) having a heparanase inhibitory activity, heparanase inhibitory activity, cancer metastasis inhibitory activity and angiogenesis inhibitory activity useful as an anticancer agent.

Formula 1

In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group.

Referring to the present invention in more detail as follows.

The isolate and identification process of the new strain fungal acremonium MT70646 (KCTC 8973P) according to the present invention is as follows.

[Isolation of New Microorganisms]

Sterilized into the soil sample was air dried for 1 g of physiological saline 10 ㎖ then plated for 30 minutes and then stirred 10 ^-2 to 10 ^-4 Strauss-fold diluted with 0.1 ㎖ Potato deck by agar (potato dextrose agar) medium at 25 ℃ 7 Incubate for 10 days to separate pure colonies.

[Identification of New Microorganisms]

1) Morphology and Culture Characteristics

The isolated strain MT70646 is a strain showing typical mold characteristics, and the culture characteristics are as follows. When cultured in potato dextrose agar medium, the color of mycelia grew to white at first, and gradually became pale orange. After 14 days of cultivation, the diameter of mycelium was about 3.5 cm. In culture for more than 14 days, the mycelial growth medium becomes hard, and the white orange mycelia are formed in the old orange mycelia. As a result of microscopic observation, the hyphae were long and entangled in a thread shape having a thickness of 2.0 to 2.5 μm, and these dozens of mycelia form bundles of various sizes, among which large bundles are connected to each other in a mesh shape. In small-sized bundles, each single mycelium branched in several directions. The color of the medium changed from early pale yellow to brown at the end of the culture.

2) Physiological Characteristics

The growth of MT70646 reached its highest on day 6 of culture in liquid medium and the production of new compounds was maximized on 5 days of culture. The optimum incubation temperature for production was 26 ℃ and when the incubation at pH 6.8 began to drop to about pH 5.5, slightly acidic after 3 days and gradually increased over time to pH 7.5 ~ 8.0 at the end of the culture. The production conditions of the new compound is essential to incubation conditions of aeration 1.0 vvm, stirring speed 300 rpm, temperature 26 ℃.

3) Identification and Naming of Mycobacteria Microorganisms MT70646

As a result of analyzing the morphological, cultural and physiological characteristics of the new strain microorganism MT70646 [see Table 1], the isolated strain is a microorganism belonging to the genus Acremonium sp. Medically Important Fungi (1995, ASM press, Washington, DC) and Compendium of Soil Fungi (1980, Academic Press, London).

Accordingly, the new strain isolated from the present invention was named MT70646 in the genus Acremoniium, and was deposited on November 5, 1999 by the Gene Bank in the Biotechnology Research Institute, a depository institution for Korean patent strains, and was given accession number KCTC 8973P.

[Cultivation of microbial microorganisms]

To cultivate fungal strain MT70646 (KCTC 8973P), a medium containing a nutrient source commonly used by microorganisms is prepared. That is, the carbon source uses glucose, fructose and the like, and the nitrogen source uses peptone, tryptone and the like. It is preferable to add magnesium sulfate and other inorganic salts as needed. As the culture method, a liquid immersion culture method under aerobic conditions at 23 to 28 ° C is suitable.

In other words, as a medium and production medium, yeast extract (0.3%), malt extract (0.3%), peptone (0.5%), glucose (2%), magnesium sulfate, 7H ₂ O (0.05%) A medium containing potassium dihydrogen phosphate (0.1%) is used. 1 ml Erlenmeyer flasks were autoclaved at 200 ml for 20 minutes at 121 ° C., and then inoculated with platinum-strained MT70646 (KCTC 8973P) with platinum, followed by shaking culture at 25 ° C. for 3 days. It is cultured. A 15 L fermentor containing 10 L of production medium was autoclaved at 121 ° C for 1 hour, and then inoculated with 600 ml of the above culture medium, followed by stirring at a speed of 300 rpm at 26 ° C and aerobic conditions under aeration. Incubate for 7 days at.

[Isolation and Purification of Novel Compounds Produced from Mold MT70646 (KCTC 8973P)]

The culture solution of Acremonium MT70646 (KCTC 8973P) was extracted with a butanol solvent, concentrated using a reduced pressure evaporator using a reduced pressure dryer, and then separated by adsorption chromatography, gel filtration chromatography, and high pressure liquid chromatography. As a result of purification, a novel compound represented by Chemical Formula 1 is obtained.

Accordingly, in the present invention, for convenience, the compound represented by the following Chemical Formula 1 is abbreviated as follows: R ₁ = H, R ₂ = (CH ₂ ) ₁₄ CH ₃ , R ₃ = H compound, that is represented by the following Chemical Formula 1a 6- [4-carboxy-3-hydroxy-5-methyl-phenoxycarbonyl) -3-hydroxy-5-pentadecyl-phenoxyl] -3,4,5-trihydroxy-tetrahydro-pyran 2-carboxylic acid is abbreviated as "CRM646-A". And a compound wherein R ₁ = CH ₃ , R ₂ = (CH ₂ ) ₁₄ CH ₃ , R ₃ = H, that is, 6- [4-carboxy-3-hydroxy-5-methyl-phenoxyca represented by the following Chemical Formula 1b: Lvonyl) -3-hydroxy-5-pentadecyl-phenoxyl] -3,4,5-trihydroxy-tetrahydro-pyran-2-carboxy methyl ester is abbreviated as "CRM646-B".

As a result of analyzing the specific structure of each compound represented by Formula 1, the following conclusions were obtained.

The compound represented by Chemical Formula 1a (CRM646-A) has a white powder property and is well soluble in solvents such as methanol, ethanol, butanol, and dimethylsulfoxide (DMSO), but is insoluble in water, hexane, chloroform, and the like. Ultraviolet spectrophotometric spectral analysis showed maximum absorption bands at 214, 260 and 305 nm, and high resolution mass spectrometry (HRFAB-MS) showed a molecular weight of 690 and a molecular formula of C ₃₆ H ₅₀ O ₁₃ . In addition, the results of the far-infrared (IR) spectrum analysis are shown in Table 2, and the results of analysis of the proton ( ¹ H) and carbon ( ¹³ C) nuclear magnetic resonance spectra are shown in Table 3 and accompanying Figures 1 and 2, and HMBC nuclear magnetic field. The resonance spectrum analysis results are shown in FIG. 3. As a result, it was confirmed that CRM646-A is a novel compound having a structure as shown in Formula 1a, and TPI-3 and 4 having inhibitory activity against phosphodiesterase among the known compounds have the same molecular weight and structural formula It is similar, but it is a structurally distinct compound because there is a difference in the methyl group located in one ring of CRM646-A, and the length and position of aliphatic carbon bonded to the other ring, and the type of sugar (glucuronic acid). Could know. Therefore, CRM646-A must be a novel bioactive substance having heparanase and heparanase inhibitory activity.

In addition, the compound represented by Chemical Formula 1b (CRM646-B) as the derivative of CRM646-A described above was found to have a molecular weight of 704 and a molecular formula of C ₃₇ H ₅₂ O ₁₃ as a result of high resolution mass spectrometry. Other physical and chemical properties are almost the same as those of CRM646-A. Far infrared (IR) spectrum analysis results are shown in Table 2, and proton ( ¹ H) and carbon ( ¹³ C) nuclear magnetic resonance spectrum analysis results are shown in Table 3 and Figures 4 and 5, respectively. As a result, peaks that were not identified in CRM646-A were observed at 3.66 ppm in the proton resonance spectrum and 51.95 ppm in the carbon resonance spectrum, indicating that a methoxy (OCH ₃ ) peak exists. This was found to be bound to the carboxyl end of C-6 of glucuronic acid present in the compound of CRM646-A. Therefore, CRM646-B is a modification of CRM646-A, a novel compound, which must also be a novel bioactive substance.

On the other hand, the present invention includes a heparanase activity inhibitor, heparanase activity inhibitor, cancer metastasis inhibitor and angiogenesis inhibitor containing the novel compound represented by the formula (1) as an active ingredient. That is, it was found for the first time that heparanase and heparanase inhibitory activity against each of the compounds represented by the formula (1) separated and purified from the fungal strain Acremonium genus MT70646 (KCTC 8973P), and each of these compounds was effective. An anticancer drug composition containing the ingredient was developed.

In addition to the active ingredient represented by Formula 1, the composition according to the present invention may be a tablet, powder, granule, capsule, using a pharmaceutically acceptable carrier or excipient that can be easily understood by those skilled in the art. It may be formulated into a unit dosage form or a multiple dosage form for suspension, emulsion or parenteral administration.

The effective dose of the active ingredient represented by Formula 1 may vary depending on the age, physical condition, weight, etc. of the patient, but is generally administered within the range of 1 to 100 mg / kg (weight) per day. In addition, within the effective daily dosage range divided into once a day or several times a day.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by Examples.

Example 1 Culture of New Strain Acremonidium MT70646 (KCTC 8973P)

Yeast extract (0.3%), malt extract (0.3%), peptone (0.5%), glucose (2%), magnesium sulfate, 7H ₂ O (0.05%), potassium dihydro as species and production medium of mold MT70646 A medium containing gen phosphate (0.1%) was used. After autoclaving a 1 L Erlenmeyer flask containing 200 ml of seed medium at 121 ° C. for 20 minutes, the MT70646 strain, which was incubated in potato dextrose or YM medium, was inoculated with platinum and shaken at 26 ° C. for 4 days. Species culture of the incubator was used. A 15 L fermentor containing 10 L of production medium was autoclaved at 121 ° C. for 1 hour and then inoculated with 200 ml of the above culture medium, followed by stirring at a speed of 300 rpm at 26 ° C. under aeration of 1.0 vvm. Incubated for 7 days in aerobic conditions.

Example 2 Isolation and Purification of CRM646-A and CRM646-B

After adding the same amount of acetone to the culture solution of the new strain Acremoniium (KCTC 8973P), the extract was concentrated under reduced pressure for 3 hours, suspended in appropriate water, and extracted three times with the same amount of butanol, followed by CRM646-A and-. A crude extract containing B was obtained. The crude extract was adsorbed onto silica gel and subjected to silica gel column chromatography. At this time, the heparinase active fraction was eluted with chloroform / methanol (20/1, v / v) stepwise by increasing the methanol concentration. Only the eluted active fractions were concentrated under reduced pressure, and then 60% acetonitrile was subjected to RP-18 column chromatography using a developing solvent. The active fraction was re-concentrated, dissolved in a small amount of 50% acetonitrile, and subjected to Sephadex LH-20 column chromatography using the same solvent as the developing solvent. The active fractions were collected and concentrated to give a pale yellow fraction, which was then subjected to high pressure liquid chromatography (column: J'sphere ODS-H80, solvent: 80% acetonitrile containing 0.05% trichloroacetic acid, flow rate: 5 ml). / Min, detection: 210 nm), CRM646-A for the residence time of 17 minutes and CRM646-B for 26 components were purely separated, and each of them was concentrated under reduced pressure to obtain a white powder.

Example 3 Measurement of Heparinase Inhibitory Activity

3 μl of the sample solution to be assayed is added to 17 μl of the buffer solution containing 10 ng of heparin, 0.2 units of heparanase are added, and the mixture is left at room temperature for 15 minutes. Again, 25 μl of antithrombin III solution was added and reacted for 2 minutes at room temperature, followed by 25 μl of factor VIIa solution. One minute after the addition, 25 μl of Pecta®a substrate was added to react for 5 to 10 minutes, and 25 μl of glacial acetic acid was added to stop the reaction, and then absorbance was measured at 410 nm. Enzyme inhibition rate was calculated by the following equation (1), IC ₅₀ value was determined by the concentration of the inhibitor to the inhibition rate of the enzyme activity 50%.

In Equation 1 above:

A represents the absorbance after the reaction of the sample without the inhibitor, B represents the absorbance after the reaction of the sample without the enzyme solution, and C represents the absorbance after the reaction of the sample with the inhibitor.

Separate new compounds CRM646 and CRM646-A-B The result of measuring the concentration (IC ₅₀₎ for inhibiting the activity of the enzyme 50% HEPA Lena found to be 3 μM and 25 μM, respectively. Suramin, on the other hand, showed 5 μM in this experiment. At this time, heparanase was isolated from Flavobacterium heparinum and was used from Sigma Co.

Example 4 Measurement of Heparanase Inhibitory Activity

Tritium ⁽³ H) the heparan only Nu of 5mM acetyl labor sulfate 90 pmol yen (N-acetylmannosmine) and 0.1mg of bovine serum albumin, 0.05M sodium acetate (Na-acetate) which contains (BSA) labeled with Add 17 µl of buffer solution (pH 5.1), add 3 µl of the sample solution to be assayed, add 10 ng of human platelet-derived heparanase, and then react at 37 ° C for 30 minutes. After the reaction, the reaction mixture solution was passed through a column filled with HRG-Sepharose beads to elute only heparan sulfate decomposed by heparanase to measure radioactivity. The enzyme inhibition rate of the sample was calculated as in Equation 2 below, and the IC ₅₀ value was determined as the concentration of the inhibitor whose inhibition rate of the enzyme activity reaches 50%.

In Equation 2 above:

A represents the radioactivity after the reaction of the sample without the inhibitor, B represents the radioactivity after the reaction of the sample without the enzyme solution, and C represents the radioactivity after the reaction of the sample with the inhibitor.

When the new compounds CRM646-A and CRM646-B separated the inhibitory activity of heparanase enzyme by 50% (IC ₅₀ ), both compounds were found to be 10 μM.

Example 5 Measurement of Inhibitory Activity of Metastasis (Cancer Infiltration) of B16 Melanoma Cells through Synthetic Basement Membrane

Inflate the Biocoat chamber with serum-free medium for 30 minutes, place the chamber in each well containing 600 μl of HT 1080 conditioned medium and add 0.5% albumin (BSA). Melanoma cells were placed in DMEM medium at a concentration of 4.5 × 10 ³ and 450 μl each. 50 μl of sample solution was added thereto, and the cells were incubated for 22 to 24 hours at 37 ° C., and the cells were immobilized with methanol and stained with 5% crystal violet. It was.

As shown in Table 4, CRM646-A showed relatively weak activity, but CRM646-B showed an IC ₅₀ value of about 2 ㎍ / mL and showed almost perfect inhibitory activity at a concentration of 10 ㎍ / mL. have.

Example 6: Determination of angiogenesis inhibitory activity

After dissolving Matrigel on ice, 40 μl of each was placed in each well of a cold 96-well incubator and left at 37 ° C. for 30 minutes to solidify the Matrigel and HUVEC, a human Umbilical Vein Endothelial Cell, was 180. 2 × 10 ⁴ μl into each well of the solidified Matrigel was incubated for 18 hours in a CO ₂ incubator at 37 ℃ and observed the neovascular formation.

As shown in Table 5, CRM646-B had more potent antiangiogenic activity than CRM646-A and showed about 60% inhibition at a concentration of 10 μg / ml.

Example 7 : Toxicity Test

In order to determine the toxicity of the novel compound represented by Formula 1 according to the present invention, the administration of the new compound intraperitoneally to 10 mice for 14 days at a 300 mg / kg dose was confirmed whether the behavior abnormality and survival . As a result, the mice administered 300 mg / kg did not observe any abnormal behavior during the administration period and survived until the last day. In addition, there was no statistically significant difference in the behavioral observation compared with the mice that did not receive the drug. In view of the above results, the toxic dose in mice treated with the novel compounds of the present invention is determined to be 300 mg / kg or more.

Example 8 Preparation of Tablets

10 g of active ingredients

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

Total amount 100 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet was 100 mg, of which the active ingredient content was 10 mg.

Example 9 Preparation of Powder

10 g of active ingredients

50 g of corn starch

40 g of carboxy cellulose

Total amount 100 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was added to the 5 times hard capsule to prepare a capsule.

Example 10 Preparation of Injectables

1 g of active ingredients

0.6 g sodium chloride

0.1 g of ascorbic acid

Water for injection

Total amount 100 ml

A 100 ml solution was obtained with the same composition as above. This solution contained 10 mg of active ingredient, which was placed in an ampoule and heat sterilized at 20 ° C. for 30 minutes.

As described above, the novel compound represented by the formula (1) isolated and purified from the new strain Acremonium genus MT70646 (KCTC 8973P) according to the present invention is excellent in inhibiting the activity of heparanase or heparanase.

Therefore, the composition comprising the novel compound represented by the formula (1) isolated from the new strain Acremoniium genus MT70646 (KCTC 8973P) as an active ingredient inhibits heparanase activity, heparanase activity inhibition, cancer metastasis inhibition and angiogenesis inhibition Very effective at

Claims (8)

Acremoniium genus MT70646 (KCTC 8973P), characterized by producing anti-cancer active substances. A compound characterized by the following formula (1). Formula 1 In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group. The compound of claim 2, wherein the compound is represented by the following Chemical Formula 1a. Formula 1a The compound according to claim 2, wherein the compound is represented by the following Chemical Formula 1b. Formula 1b Heparinase inhibitors, characterized in that the compound represented by the following formula (1) is contained as an active ingredient. Formula 1 In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group. Heparanase inhibitors, characterized in that the compound represented by the following formula (1) is contained as an active ingredient. Formula 1 In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group. Cancer metastasis inhibitor, characterized in that the compound represented by the following formula (1) is contained as an active ingredient. Formula 1 In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group. An angiogenesis inhibitor, characterized in that the compound represented by the following formula (1) is contained as an active ingredient. Formula 1 In Formula 1, R ₁ represents a hydrogen atom, a halogen atom, or a C _{1 to} C ₆ alkyl group; R ₂ represents a C ₁ to C ₂₀ alkyl group; R ₃ represents a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group.